A major goal of restorative dentistry is the replacement of carious tooth structure with materials that closely resemble that natural mineralized tissue, while preserving the vitality of the pulpo-dentinal complex. Pulp tissue exhibits an intrinsic defense mechanism against insult or injury culminating in the differentiation of new odontoblasts and the deposition of tertiary dentin. While the formation of reactionary or reparative dentin by the pulp tissue is an inherent response to injury or insult, it is not routinely evoked in modern clinical treatments. Our goal is to develop a new dental material that can be applied to the floor of a cavity to evoke this biologically favorable response of self-healing. Studies suggest that cytokines of the TGF-beta family, in particular bone morphogenetic proteins, as well as other, recently identified mineralized matrix molecules (e.g. bone sialoprotein, amelogenin, dentin sialophosphoprotein, dentin matrix protein 1) are capable of inducing proliferative and/or phenotypic changes in dental pulp cells that are comparable to those occurring during the reparative process. Our hypothesis is that dental material containing dentin-inducing molecules when placed on the floor of a cavity preparation will induce tertiary dentin formation by the underlying dental pulp tissue in a predictable manner. To test this hypothesis, we propose the following four specific aims: Specific Aim 1 will use newly developed human dental cell lines to screen potential dentin-inductive molecules in a novel DSPP gene activation assay. Specific Aim 2 will determine the mechanism of action of identified inductive molecules. Specific Aim 3 will characterize the optimal concentration and time-course of promising dentin inductive molecules on the phenotypic changes in human dental pulp and odontoblast cells in vitro. Specific Aim 4 will determine the parameters of transdentinal movement of the dentin inductive molecules in combination with our cyclodextrin carrier system in vitro. While our goal and hypothesis have not changed in this continuation proposal, we have broadened our scope to a more general survey of suitable dentin-inductive molecules to be used in combination with our novel restorative system. The ideal dentin-inductive molecule should be readily available, should have prolonged biological activity either alone or in complex with carrier molecules like cyclodextrins, and have excellent biocompatibility. We remain committed to see transdentinal tertiary dentin induction becoming an integral part of the restorative dental armentarium.